WO2022191550A1 - ＩｇＥ Ｆｃ 수용체의 알파 서브유닛의 세포외 도메인을 포함하는 융합단백질의 제형 - Google Patents

ＩｇＥ Ｆｃ 수용체의 알파 서브유닛의 세포외 도메인을 포함하는 융합단백질의 제형 Download PDF

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WO2022191550A1
WO2022191550A1 PCT/KR2022/003207 KR2022003207W WO2022191550A1 WO 2022191550 A1 WO2022191550 A1 WO 2022191550A1 KR 2022003207 W KR2022003207 W KR 2022003207W WO 2022191550 A1 WO2022191550 A1 WO 2022191550A1
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formulation
aqueous pharmaceutical
pharmaceutical formulation
concentration
ige
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PCT/KR2022/003207
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English (en)
French (fr)
Korean (ko)
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장명호
조영규
권순탁
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(주)지아이이노베이션
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Priority to US18/549,558 priority Critical patent/US20240148829A1/en
Priority to AU2022232566A priority patent/AU2022232566A1/en
Priority to JP2023555346A priority patent/JP2024509937A/ja
Priority to PE2023002481A priority patent/PE20240120A1/es
Priority to CN202280016419.9A priority patent/CN116963721A/zh
Priority to EP22767446.2A priority patent/EP4306103A1/en
Priority to CA3211978A priority patent/CA3211978A1/en
Priority to MX2023010457A priority patent/MX2023010457A/es
Priority to IL305512A priority patent/IL305512A/en
Publication of WO2022191550A1 publication Critical patent/WO2022191550A1/ko

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/177—Receptors; Cell surface antigens; Cell surface determinants
    • A61K38/1774—Immunoglobulin superfamily (e.g. CD2, CD4, CD8, ICAM molecules, B7 molecules, Fc-receptors, MHC-molecules)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/08—Solutions
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/06—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite
    • A61K47/08—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite containing oxygen, e.g. ethers, acetals, ketones, quinones, aldehydes, peroxides
    • A61K47/10—Alcohols; Phenols; Salts thereof, e.g. glycerol; Polyethylene glycols [PEG]; Poloxamers; PEG/POE alkyl ethers
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/06—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite
    • A61K47/16—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite containing nitrogen, e.g. nitro-, nitroso-, azo-compounds, nitriles, cyanates
    • A61K47/18—Amines; Amides; Ureas; Quaternary ammonium compounds; Amino acids; Oligopeptides having up to five amino acids
    • A61K47/183—Amino acids, e.g. glycine, EDTA or aspartame
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/06—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite
    • A61K47/20—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite containing sulfur, e.g. dimethyl sulfoxide [DMSO], docusate, sodium lauryl sulfate or aminosulfonic acids
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/06—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite
    • A61K47/22—Heterocyclic compounds, e.g. ascorbic acid, tocopherol or pyrrolidones
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/06—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite
    • A61K47/26—Carbohydrates, e.g. sugar alcohols, amino sugars, nucleic acids, mono-, di- or oligo-saccharides; Derivatives thereof, e.g. polysorbates, sorbitan fatty acid esters or glycyrrhizin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/0012—Galenical forms characterised by the site of application
    • A61K9/0019—Injectable compositions; Intramuscular, intravenous, arterial, subcutaneous administration; Compositions to be administered through the skin in an invasive manner
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/0012—Galenical forms characterised by the site of application
    • A61K9/0019—Injectable compositions; Intramuscular, intravenous, arterial, subcutaneous administration; Compositions to be administered through the skin in an invasive manner
    • A61K9/0021—Intradermal administration, e.g. through microneedle arrays, needleless injectors
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/08—Antiallergic agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/70503—Immunoglobulin superfamily
    • C07K14/70535—Fc-receptors, e.g. CD16, CD32, CD64 (CD2314/705F)
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/30—Non-immunoglobulin-derived peptide or protein having an immunoglobulin constant or Fc region, or a fragment thereof, attached thereto
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/33—Fusion polypeptide fusions for targeting to specific cell types, e.g. tissue specific targeting, targeting of a bacterial subspecies

Definitions

  • the present invention relates to a formulation optimized for a fusion protein dimer comprising the extracellular domain of the alpha subunit of an IgE Fc receptor.
  • Allergic diseases such as asthma, allergic rhinitis, atopic dermatitis, and food allergy are rapidly increasing in industrialized and westernized modern society, and the occurrence of anaphylaxis, a severe allergic disease, is also increasing.
  • These chronic immune diseases greatly reduce the quality of individual life, and the social and economic costs are also rapidly increasing.
  • IgE immunoglobulin E
  • IgE immunoglobulin E
  • Fc ⁇ RI high-affinity IgE Fc receptor
  • mast cells or basophilic granulocytes release chemical mediators such as histamine, leukotriene, prostaglandin, bradykinin and platelet-activating factor.
  • Allergic symptoms are caused by the release of these chemical mediators.
  • allergic diseases may be exacerbated by the binding between IgE and Fc ⁇ RI.
  • immunoglobulin compositions capable of binding IgE and Fc ⁇ RIIb with high affinity and inhibiting IgE-expressing cells are being studied (KR 10-1783272B1). Such compositions have been reported to be useful for treating IgE-mediated disorders, including allergies and asthma.
  • Omalizumab (trade name: Xolair), which targets the Fc portion of an IgE antibody, has been developed and is being used as a treatment for intractable severe asthma and intractable urticaria.
  • the cost burden is high because high doses must be administered in order to maintain the effect, and there are side effects such as angioedema and anaphylactic reaction.
  • side effects such as angioedema and anaphylactic reaction.
  • post-marketing results have reported allergic granulomatous vasculitis and idiopathic severe thrombocytopenia, the need for developing therapeutic agents that can effectively treat allergic diseases without side effects is increasing.
  • a fusion protein including the extracellular domain of the alpha subunit of the IgE Fc receptor was developed, and it was confirmed that the fusion protein can treat allergic diseases ( KR 10-2038675B1).
  • the fusion protein can treat allergic diseases ( KR 10-2038675B1).
  • the present invention was completed by developing a high-concentration aqueous pharmaceutical formulation optimized for subcutaneous injection application in order to commercialize the fusion protein dimer.
  • the present invention provides an aqueous pharmaceutical formulation comprising a fusion protein dimer comprising the extracellular domain (Fc ⁇ RI ⁇ ECD) of the alpha subunit of the IgE Fc receptor, and having a pH of 6.0 to 7.0.
  • an aqueous pharmaceutical formulation comprising a fusion protein dimer including the extracellular domain (Fc ⁇ RI ⁇ ECD) of the alpha subunit of the IgE Fc receptor according to the present invention and having a pH of 6.0 to 7.0 has excellent stability. Furthermore, the formulation according to the present invention contains the fusion protein dimer at a high concentration, can be administered quickly and conveniently by subcutaneous injection, has excellent stability, and can increase the storage period by reducing the formation of aggregates. Therefore, the formulation comprising the fusion protein dimer including the extracellular domain of the alpha subunit of the IgE Fc receptor (Fc ⁇ RI ⁇ ECD) can be usefully used as an injection for treating allergic diseases.
  • 3 is a photograph of samples (#1 to #6) subjected to stress for 14 days at 35°C and 200 rpm.
  • 5 is a graph showing main peak areas of samples according to each stress condition.
  • 6 is a graph showing aggregation areas of samples according to each stress condition.
  • FIG 9 is a graph showing a force/path diagram. At this time, the extrusion speed was performed at 190 mm/min.
  • 10 is a graph showing main peak areas of samples #1 and #2 according to each stress condition.
  • aqueous pharmaceutical formulation comprising a fusion protein dimer comprising the extracellular domain (Fc ⁇ RI ⁇ ECD) of the alpha subunit of the IgE Fc receptor.
  • the pH of the aqueous pharmaceutical formulation may be in the range of 6.0 to 7.0, 6.1 to 7.0, 6.2 to 6.9, 6.3 to 6.8, 6.4 to 6.8, or 6.4 to 6.6.
  • the term "pharmaceutical formulation” refers to a formulation that is in a form that enables the biological activity of the active ingredient to be clearly effective, and does not contain ingredients that cause side effects to the subject to which the formulation is administered.
  • the term "individual” may be a mammal such as a human, dog, cow, horse, pig, sheep, goat, cat, mouse, rabbit, or rat, preferably a human, dog or cat.
  • aqueous pharmaceutical formulation refers to a pharmaceutical formulation using a suitable aqueous solvent such as water or an aqueous/oily mixture (eg, a water alcohol mixture).
  • a suitable aqueous solvent such as water or an aqueous/oily mixture (eg, a water alcohol mixture).
  • the formulation is capable of maintaining stability, such as chemical or physical stability, biological activity.
  • the term “stability” refers to the property of maintaining a constant state, and generally includes minimizing degradation, denaturation, aggregation or unfolding of biologically active substances, such as proteins, peptides, or bioactive macromolecules. related
  • the formulation may be a liquid formulation.
  • Liquid formulations are aqueous solutions or suspensions and can remain stable during storage at room temperature, refrigeration (eg, 2°C to 8°C) or frozen (eg, -20°C or -70°C).
  • aqueous pharmaceutical formulation of the present invention may be administered parenterally.
  • parenteral administration may be performed by subcutaneous administration, intravenous administration, mucosal administration, intramuscular administration, or the like.
  • the formulation is preferably administered by subcutaneous injection.
  • the fusion protein dimer in the aqueous pharmaceutical formulation may be included in a high concentration, specifically 50 mg/mL or more, 60 mg/mL or more, or 70 mg/mL or more.
  • the concentration of the fusion protein dimer is not limited thereto, but 50 mg/mL to 150 mg/mL, 50 mg/mL to 130 mg/mL, 50 mg/mL to 120 mg/mL , 50 mg/mL to 110 mg/mL, 50 mg/mL to 100 mg/mL, 50 mg/mL to 90 mg/mL, 50 mg/mL to 80 mg/mL, 60 mg/mL to 150 mg/mL , 60 mg/mL to 130 mg/mL, 60 mg/mL to 120 mg/mL, 60 mg/mL to 110 mg/mL, 60 mg/mL to 100 mg/mL, 60 mg/mL to 90 mg/mL , 60 mg/mL to 80 mg/mL, 70 mg/
  • the aqueous pharmaceutical formulation may further include a buffer and a stabilizer.
  • the buffer may be histidine.
  • the histidine may be present in a concentration of 10 mM to 100 mM in the formulation.
  • the histidine as a buffer is 10 mM to 90 mM, 10 mM to 80 mM, 10 mM to 70 mM, 10 mM to 60 mM, 20 mM to 90 mM, 20 mM to 80 mM in the formulation.
  • the histidine buffer is not limited thereto, but may include a solution of histidine chloride, histidine acetate, histidine phosphate, and histidine sulfate.
  • the pH of the histidine buffer or histidine-HCl buffer may be in the range of 5.5 to 7.5, in the range of 6.0 to 7.0, in the range of 6.1 to 7.0, in the range of 6.2 to 6.9, in the range of 6.3 to 6.8, in the range of 6.4 to 6.8 or in the range of 6.4 to 6.6, preferably may be pH 6.5.
  • the stabilizer may be proline.
  • the proline may be present in a concentration of 200 mM to 300 mM in the formulation.
  • the proline is 200 mM to 290 mM, 200 mM to 280 mM, 200 mM to 270 mM, 200 mM to 260 mM, 210 mM to 290 mM, 210 mM to 280 mM, 210 in the formulation mM to 270 mM, 210 mM to 260 mM, 220 mM to 290 mM, 220 mM to 280 mM, 220 mM to 270 mM, 220 mM to 260 mM, 230 mM to 290 mM, 230 mM to 280 mM, 230 mM to 270 mM, 230 mM to 280 mM, 230 mM to 270 mM, 230 mM to 280 mM
  • the aqueous pharmaceutical formulation of the present invention may further contain an antioxidant.
  • the antioxidant may be methionine.
  • the methionine may be present in a concentration of 10 mg/mL to 30 mg/mL in the formulation.
  • the methionine is 10 mg/mL to 28 mg/mL, 10 mg/mL to 26 mg/mL, 10 mg/mL to 24 mg/mL, 10 mg/mL to 22 mg in the formulation.
  • the antioxidant is an agent that inhibits the oxidation of other molecules, and can reduce the formation of aggregates in the formulation by removing oxygen.
  • the aqueous pharmaceutical formulation may further comprise a surfactant.
  • the surfactant is an agent that lowers the surface tension of the liquid, and can control the formation of soluble and insoluble aggregates.
  • the surfactant may be a non-ionic surfactant, including but not limited to polysorbate 20, polysorbate 28, polysorbate 40, polysorbate 60, polysorbate 65, polysorbate 80, polysorbate polysorbates such as sorbate 81 and polysorbate 85; poloxamers such as poloxamer 181, poloxamer 188, poloxamer 407; or polyethylene glycol (PEG).
  • the surfactant may be a poloxamer.
  • the poloxamer may be poloxamer 188 (poloxamer 188).
  • the poloxamer 188 may be present in a concentration of 0.01 w/v% to 1 w/v% in the formulation.
  • the poloxamer 188 is 0.02 w/v% to 0.8 w/v%, 0.02 w/v% to 0.6 w/v%, 0.02 w/v% to 0.4 w/v% in the formulation.
  • a fusion protein dimer comprising Fc ⁇ RI ⁇ ECD at a concentration of 50 mg/mL to 150 mg/mL; ii) histidine at a concentration of 10 mM to 100 mM; iii) proline at a concentration of 200 mM to 300 mM; iv) methionine at a concentration of 10 mg/mL to 30 mg/mL; and v) poloxamer 188 at a concentration of 0.01 w/v% to 1 w/v%.
  • the pH of the aqueous pharmaceutical formulation may be in the range of 6.0 to 7.0, 6.1 to 7.0, 6.2 to 6.9, 6.3 to 6.8, 6.4 to 6.8, or 6.4 to 6.6. Preferably, it may be pH 6.5.
  • the aqueous pharmaceutical formulation comprises: i) a fusion protein dimer comprising Fc ⁇ RI ⁇ ECD at a concentration of 75 mg/mL; ii) histidine at a concentration of 55 mM; iii) proline at a concentration of 250 mM; iv) methionine at a concentration of 20 mg/mL; and v) poloxamer 188 at a concentration of 0.1 w/v%.
  • the aqueous pharmaceutical formulation may be stored in a container selected from the group consisting of vials, cartridges, syringes and autoinjectors.
  • the container in which the formulation is stored may be stored at room temperature, 2°C to 8°C, or 25°C to 40°C until administered to a subject in need of treatment.
  • the formulation is not limited thereto, but can be administered parenterally, such as subcutaneously, intravenously, mucosally, intramuscularly, intraperitoneally, using an 18G to 32G needle in a volume of 5 mL or less, 3 mL or less, or 2 mL or less. have. Preferably, it can be administered subcutaneously using a 27G needle in a volume of 1 mL or less.
  • Fusion protein comprising the extracellular domain of the alpha subunit of an IgE Fc receptor
  • the fusion protein dimer described above is as follows. Specifically, the dimer comprises two monomers comprising the extracellular domain (Fc ⁇ RI ⁇ ECD) of the alpha subunit of the IgE Fc receptor.
  • the monomer may include a modified Fc region, and the modified Fc region and Fc ⁇ RI ⁇ ECD may be linked through a hinge of an IgD antibody.
  • IgE refers to an antibody protein known as immunoglobulin E. IgE has affinity for mast cells and basophils in blood. In addition, when an IgE antibody and its corresponding antigen (allergen) react, an inflammatory reaction is caused. In addition, IgE is known as an antibody causing anaphylaxis, which appears when mast cells or basophils are suddenly secreted.
  • IgE Fc receptor is also called Fc ⁇ receptor, and binds to the Fc portion of IgE.
  • Fc ⁇ RI Fc ⁇ receptor I
  • Fc ⁇ RII Fc ⁇ receptor II
  • Fc ⁇ RI Fc ⁇ receptor I
  • Fc ⁇ RII Fc ⁇ receptor II
  • Fc ⁇ RI is expressed in mast cells and basophils.
  • an Fc ⁇ RI-bound IgE antibody is cross-linked by a multivalent antigen, degranulation occurs in mast cells or basophils, releasing various chemical transmitters including histamine. This release results in an immediate allergic reaction.
  • the Fc ⁇ RI is a membrane protein composed of one ⁇ chain, one ⁇ chain, and two ⁇ chains bonded to disulfide.
  • the part to which IgE binds is the ⁇ chain (Fc ⁇ RI ⁇ )
  • Fc ⁇ RI ⁇ has a size of about 60 kDa, and is composed of a hydrophobic domain existing in the cell membrane and a hydrophilic domain existing outside the cell membrane.
  • IgE binds to the extracellular domain of the ⁇ chain.
  • the alpha subunit of the IgE Fc receptor may have the amino acid sequence described in NP_001992.1.
  • the extracellular domain (Fc ⁇ RI ⁇ ECD) of the alpha subunit of the IgE Fc receptor may have the amino acid sequence of SEQ ID NO: 1.
  • the extracellular domain of the alpha subunit of the IgE Fc receptor may be a fragment or variant of the extracellular domain of the alpha subunit of the IgE Fc receptor as long as it can bind to IgE.
  • the mutant may be performed by substituting, deleting or adding one or more proteins in the wild-type Fc ⁇ RI ⁇ ECD (extracellular domain) as long as the function of the ⁇ chain of Fc ⁇ RI is not altered.
  • These various proteins or peptides may be at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 1.
  • the Fc ⁇ RI ⁇ ECD of SEQ ID NO: 1 may be encoded by a polynucleotide having the sequence of SEQ ID NO: 5.
  • modified Fc region refers to a region in which some of the Fc portion of an antibody is modified.
  • Fc region includes the heavy chain constant region 2 (CH2) and heavy chain constant region 3 (CH3) of an immunoglobulin, and does not include the variable region and light chain constant region 1 (CH1) of the heavy and light chains of an immunoglobulin.
  • protein that does not In particular, the modified Fc region means that some amino acids in the Fc region are substituted or prepared by combining different types of Fc regions.
  • the modified Fc region may have the amino acid sequence of SEQ ID NO: 2.
  • the modified Fc region of SEQ ID NO: 2 may be encoded by a polynucleotide having the sequence of SEQ ID NO: 6.
  • modified Fc region of the present invention may be a native sugar chain, an increased sugar chain compared to the native type, a decreased sugar chain compared to the native type, or a form in which the sugar chain is removed.
  • Immunoglobulin Fc sugar chains can be modified by conventional methods such as chemical methods, enzymatic methods, and genetic engineering methods using microorganisms.
  • the "modified Fc region" of the present invention does not have a binding site for Fc ⁇ R or C1q, so it may lack antibody dependent cellular cytotoxicity (ADCC) and complement dependent cytotoxicity (CDC) functions.
  • the modified Fc region and Fc ⁇ RI ⁇ ECD may be linked through the hinge of the IgD antibody.
  • the hinge of the IgD antibody consists of 64 amino acids, and may be selected from 20 to 60 consecutive amino acids, or 25 to 50 consecutive amino acids, or 30 to 40 amino acids. In one embodiment, the hinge of the IgD antibody may consist of the following 30 or 49 amino acids.
  • the hinge of the IgD antibody may be a hinge variant in which the hinge region is modified, and the hinge may include at least one cysteine. In this case, the hinge variant may be a part of the hinge sequence of the IgD antibody modified to minimize the occurrence of cleavage in the protein production process.
  • the hinge may comprise the following sequence:
  • Xaa1 may be Lys or Gly
  • Glu Xaa2 is Glu , Gly or Ser.
  • the hinge may have the amino acid sequence of SEQ ID NO: 3 and SEQ ID NO: 19, thereby minimizing the occurrence of cleavage in the protein production process.
  • Xaa3 may be Lys or Gly
  • Xaa4 may be Glu, Gly or Ser.
  • the hinge may have the amino acid sequence of SEQ ID NO: 4, thereby minimizing the occurrence of cleavage in the protein production process.
  • Thr in the hinge having the sequence of SEQ ID NO: 4, at least one of Thr may be glycosylated.
  • Thr at the 13th, 14th, 18th and 19th positions may be glycosylated.
  • all four amino acids may be glycosylated.
  • the glycosylation may be O-glycosylation.
  • the fusion protein dimer provided in the present invention may be in the form of two monomers bound to the extracellular domain of the alpha subunit of the IgE Fc receptor and the modified Fc region.
  • the fusion protein dimer may be in a form in which two identical monomers are bound by a cysteine positioned at a hinge region.
  • the polypeptide dimer may be in the form of two different monomers bonded to each other.
  • one monomer comprises the extracellular domain of the alpha subunit of the IgE Fc receptor and the other monomer comprises a fragment of the extracellular domain of the alpha subunit of the IgE Fc receptor.
  • one embodiment of the monomer may have the amino acid sequence of SEQ ID NO: 20, SEQ ID NO: 21, or SEQ ID NO: 22.
  • the fusion protein dimer provided in the present invention exhibits 10 to 100 times, 20 to 90 times, 20 to 70 times, 30 to 70 times, 40 to 70 times higher IgE binding force compared to the anti-IgE antibody omalizumab, and preferably For example, it may exhibit 70-fold higher IgE binding capacity compared to omalizumab.
  • Another aspect of the present invention provides the use of an aqueous pharmaceutical formulation comprising the fusion protein dimer for the prevention or treatment of allergic diseases.
  • allergic disease means a pathological symptom caused by an allergic reaction mediated by the activation of mast cells, such as degranulation of mast cells, and such allergic diseases include food allergy, atopic dermatitis , asthma, allergic rhinitis, allergic conjunctivitis, allergic dermatitis, allergic contact dermatitis, anaphylaxis, urticaria, pruritus, These include insect allergies, chronic idiopathic urticaria, chronic spontaneous urticaria and drug allergies.
  • the allergic disease may be an IgE-mediated disease.
  • the aqueous pharmaceutical formulation of the present invention can be administered to a subject according to a dosage regimen and dosage effective for treating an allergic disease.
  • the fusion protein dimer in the formulation of the present invention may be included in any amount (effective amount) as long as it can exhibit anti-allergic activity, and a typical effective amount is within the range of 0.001 wt% to 20.0 wt% based on the total weight of the composition. can be determined from Here, "effective amount” refers to an amount capable of inducing an anti-allergic effect. Such effective amounts can be determined empirically within the ordinary ability of one of ordinary skill in the art.
  • a preferred dosage of the formulation is in the range of 0.01 ⁇ g/kg to 10 g/kg per day, preferably 0.01 mg/kg to 1 g/kg, depending on the patient's condition, weight, sex, age, severity of the patient, and route of administration. can be a range. Administration may be performed once a day or divided into several times. Such dosages should not be construed as limiting the scope of the invention in any respect.
  • the present invention provides a method for treating or preventing an allergic disease comprising administering to an individual an aqueous pharmaceutical formulation including the fusion protein dimer.
  • the subject may be a mammal, preferably a human.
  • the administration may be administered through parenteral administration.
  • parenteral administration may be performed by subcutaneous administration, intravenous administration, mucosal administration, intramuscular administration, or the like.
  • a polypeptide in which the C terminus of the extracellular domain (Fc ⁇ RI ⁇ ECD) of the alpha subunit of the IgE Fc receptor is modified was prepared according to the method disclosed in US Pat. No. 7,867,491.
  • the extracellular domain of the ⁇ chain of Fc ⁇ RI having the amino acid sequence of SEQ ID NO: 1 and the modified immunoglobulin Fc of SEQ ID NO: 2 are respectively linked by a hinge of SEQ ID NO: 19 (Fc ⁇ RI ⁇ ECD-Fc1), SEQ ID NO: 3
  • Fc ⁇ RI ⁇ ECD-Fc2 the hinge-linked protein of SEQ ID NO: 4
  • a cassette linking the genes encoding each protein was cloned into a vector, and Fc ⁇ RI ⁇ ECD- An Fc protein expression vector was constructed. Then, each of the expression vectors was transfected into CHO cells.
  • the expression vector in which the ⁇ -2,6-sialic acid transferase gene is cloned is simultaneously transfected to obtain a cell line capable of expressing the sialic acid-added Fc ⁇ RI ⁇ ECD-Fc2ST and Fc ⁇ RI ⁇ ECD-Fc3ST proteins. prepared separately.
  • the fusion protein dimer produced from the cell line was named "GI-301".
  • the test was conducted with the aim of developing a liquid injection for subcutaneous injection that has a low viscosity that is injectable, is stable at 2°C to 8°C, and contains a GI-301 substance of about 100 mg/mL.
  • the buffer and pH conditions were optimized.
  • a total of 25 different buffers with different pH, buffer components, ionic strength and stabilizers were selected through design of experiments (DOE) and pre-screened (step 1, step 2) and final Screening (step 3) was performed.
  • DOE design of experiments
  • the denaturation temperature was determined by measuring the hydrodynamic radius of the sample by temperature in each formulation buffer. These results were used to predict formulation candidates with a low risk of protein denaturation during handling and storage. When the protein domain begins to denature, the hydrodynamic radius of the protein increases significantly.
  • the protein unfolding initiation temperature can be regarded as an indicator of protein secondary structure stability.
  • step 3 final screening (step 3):
  • the pH was adjusted to 6.0-7.4, an acceptable pH range for subcutaneous injection.
  • Appropriate buffer components were selected for each pH value to achieve sufficient buffering capacity.
  • the buffer component was selected from pharmaceutically acceptable excipients.
  • Sodium chloride was added to adjust the ionic strength of the formulation (maximum ionic strength set to isotonic). The results of the first pre-screening experiment are summarized in Table 1 below.
  • the colloidal stability of GI-301 is lower at high ionic strength and is independent of the buffer system used and formulation pH. At low ionic strength, the buffer component had the greatest effect on the colloidal stability of GI-301. The effect of pH was not significant. The best colloidal stability was found in the His/HCl buffer system, followed by Tris/HCl, sodium phosphate, and sodium citrate, followed by excellent stability. In general, when formulations in one buffer group had more acidic pH values, they exhibited low colloidal stability. Formulation sample #8 (10 mM His/HCl, pH 6.7), sample #10 (10 mM His/HCl, pH 6.0) and sample #4 (10 mM Tris/HCl, pH 7.4) showed good colloidal stability.
  • thermodynamic stability of GI-301 was similar in all formulation samples except samples #9 and #10. At commonly used drug storage temperatures, GI-301 exhibited high thermodynamic stability.
  • Example 1 Of the 25 samples tested in Example 1 above, 4 most appropriate samples were selected for the concentration loading test. In Table 5 below, four candidate formulation samples selected based on the results of Example 1 are summarized and shown.
  • Viscosity and Syringeability For each formulation sample, the push-out force was measured to evaluate the syringeability. A force/displacement measuring device was used to dispense the contents of the syringe at a specified traverse speed through an attached needle. Samples were analyzed for the following items: visual appearance, concentration by UV 280, pH, viscosity using capillary viscometer, turbidity by nephelometric measurement, osmolality, relative potency by ELISA and SE-HPLC aggregation state by
  • the injectability test confirmed that a pushing force exceeding 20 N was induced for all 100 mg/mL samples when the 30G cannula was used. With the 27G cannula, the pushing force was acceptable. When diluted to a concentration of 50 mg/mL, even a 30G cannula was found to be acceptable.
  • the final protein concentration was about 65 mg/mL.
  • a 1 mL syringe filled with 1 mL of sample was pushed through a tensioning machine, and shear-stressed material (content pushed out of the syringe) was analyzed.
  • the GI-301 protein was very stable when stored for a short period at a concentration of 100 mg/mL or less in all four formulation samples.
  • Example 2 it was confirmed that the protein could be concentrated at a high concentration in the four formulation buffers, and the injectability was also verified. As a result of analysis of the four samples, it was confirmed that there was no significant difference between them. Accordingly, a forced disintegration test was additionally performed on the four samples of Example 2 (see Table 5) using a 2R vial.
  • sample #3 (proline, histidine/HCl, pH 6.7) was found to be the most suitable buffer for the next development step. This sample was also used to perform a 150 mg/mL concentration test via cross-flow filtration.
  • a formulation containing 250 mM proline, 10 mM histidine/HCl, pH 6.7 was selected as a formulation for feasibility analysis of high concentration (150 mg/mL) through cross-flow filtration through Example 3. The goal is to obtain a concentrated protein solution (150 mg/mL).
  • buffer exchange (diafiltration each) was started in an isovolume manner.
  • the permeate drain was replaced with the target buffer supplied to the stirred vessel of the cross-flow filtration device.
  • the total supply of target buffer was 950 g.
  • the concentration of the original DS buffer was less than 1%.
  • a second concentration step was then carried out until a volume of about 110 g was reached.
  • the pH target of 6.70 was adjusted with 150 mM histidine/250 mM proline buffer. After that, the concentration step was continued.
  • the pH was fine-tuned and a concentrate was obtained.
  • the resulting solution was pre-filtered with a 1 ⁇ m filter, filtered through a 0.2 ⁇ m filter under aseptic conditions, and stored at 2° C. to 8° C. until further processing.
  • Table 8 shows the maximum pushing force generated during the measurement.
  • the 27G cannula was suitable for GI-301 at a concentration of about 100 mg/mL.
  • the 30G cannula exhibited too high a pushing force at a concentration of about 100 mg/mL.
  • Example 4 the buffer composition 250 mM proline, histidine/HCl, pH 6.7 was concentrated to a maximum concentration of about 106 mg/mL through cross-flow filtration. Therefore, forced degradation test II was conducted with a target concentration of GI-301 of 100 mg/mL.
  • a surfactant and/or antioxidant methionine was added to confirm the benefit of the additive (Table 9).
  • the 2R vials were each filled with 1.5 mL of the corresponding solution, and the samples were analyzed after stressing via heat/mechanical and light.
  • Formulation samples for forced degradation tests using surfactants and methionine are as follows:
  • Table 11 shows the sample concentrations after the forced degradation test. There was no significant difference according to stress exposure.
  • Table 12 shows the pH of the samples after the forced degradation test. There was no significant change in pH.
  • SE-HPLC is a standard method for the detection of aggregated and fragmented proteins.
  • the SEC measurement results are shown in Table 13 and FIGS. 5 to 7 .
  • the relative efficacy of GI-301 was determined according to the results of an ELISA-based efficacy assay.
  • the principle of this assay is to quantify GI-301 molecules capable of binding to human IgE.
  • Table 15 shows the turbidity measurement results. There was no clear correlation between visual appearance and turbidity measurements (compare samples #3 and #4 after 5 days storage at 35°C). A potential cause of this discrepancy may be the increased luster in the concentrated protein solution, which increases the base turbidity. As a result, the turbidity visible to the naked eye due to agglomeration can superimpose the basic turbidity. Therefore, samples were stored at 35° C. for 14 days and then analyzed by DLS.
  • sample #2 and sample #5 were found to be suitable among the formulation samples in Table 17 above.
  • Table 20 shows the tensile machine results. Pushing forces in excess of 20 N occurred in both samples when using the 27G cannula ( FIG. 9 ). Therefore, a syringe with a larger inner diameter or a larger needle size (eg, 25G) should be selected to accommodate a 100 mg/mL protein concentration.
  • Table 21 shows the concentrations of the samples after the forced degradation test. There was no significant difference after stress exposure.
  • Table 22 shows the pH of the samples after the forced degradation test. There was no significant change in pH.
  • the SEC measurement results are shown in Table 23 and FIG. 10 .
  • the osmolality results are shown in Table 24.
  • the osmolality was maintained at the same level even after stress was applied to the sample.
  • the amount of invisible particles was assessed using a fluid imaging microscope. Stressed syringes filled with water had a low particle load. This indicates that the effect of silicone syringes on particle loading is limited.
  • Samples #1 and #2 showed similar particle loading. Most of the particles observed were silicon particles, and no particles exhibited protein aggregates/precipitation.
  • sample #2 performed slightly better in SEC analysis (relative main peak area).

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PCT/KR2022/003207 2021-03-09 2022-03-07 ＩｇＥ Ｆｃ 수용체의 알파 서브유닛의 세포외 도메인을 포함하는 융합단백질의 제형 WO2022191550A1 (ko)

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US18/549,558 US20240148829A1 (en) 2021-03-09 2022-03-07 Formulation of fusion protein including extracellular domain of alpha subunit of ige fc receptor
AU2022232566A AU2022232566A1 (en) 2021-03-09 2022-03-07 Formulation of fusion protein including extracellular domain of alpha subunit of ige fc receptor
JP2023555346A JP2024509937A (ja) 2021-03-09 2022-03-07 Ｉｇｅ ｆｃ受容体のアルファサブユニットの細胞外ドメインを含む融合タンパク質の製剤
PE2023002481A PE20240120A1 (es) 2021-03-09 2022-03-07 Formulacion de proteina de fusion que incluye el dominio extracelular de la subunidad alfa del receptor ige fc
CN202280016419.9A CN116963721A (zh) 2021-03-09 2022-03-07 包含IgE Fc受体的α亚基的胞外结构域的融合蛋白的制剂
EP22767446.2A EP4306103A1 (en) 2021-03-09 2022-03-07 Formulation of fusion protein including extracellular domain of alpha subunit of ige fc receptor
CA3211978A CA3211978A1 (en) 2021-03-09 2022-03-07 Formulation of fusion protein including extracellular domain of alpha subunit of ige fc receptor
MX2023010457A MX2023010457A (es) 2021-03-09 2022-03-07 Formulacion de proteina de fusion que incluye el dominio extracelular de la subunidad alfa del receptor fc de ige.
IL305512A IL305512A (en) 2021-03-09 2022-03-09 Formulation of a fusion protein comprising the extracellular domain of the alpha subunit of the IgE FC receptor

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Citations (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US7867491B2 (en) 2007-05-30 2011-01-11 Genexine Co., Ltd. Immunoglobulin fusion proteins
KR20120103702A (ko) * 2010-01-19 2012-09-19 에프. 호프만-라 로슈 아게 단백질용 약제학적 제형물
KR101783272B1 (ko) 2008-09-17 2017-09-29 젠코어 인코포레이티드 ＩｇＥ-매개된 장애를 치료하기 위한 신규 조성물 및 방법
KR102038675B1 (ko) 2018-01-08 2019-10-30 (주)지아이이노베이션 IgE Fc 수용체의 알파 서브유닛의 세포외 도메인, 이를 포함하는 약학적 조성물 및 이의 제조방법
KR102038672B1 (ko) * 2018-01-08 2019-10-30 (주)지아이이노베이션 IgE Fc 수용체의 알파 서브유닛의 세포외 도메인을 포함하는 약학적 조성물
WO2020097141A1 (en) * 2018-11-07 2020-05-14 Merck Sharp & Dohme Corp. Stable formulations of programmed death receptor 1 (pd-1) antibodies and methods of use thereof
US20200353049A1 (en) * 2018-01-26 2020-11-12 Genentech, Inc. Compositions and methods of use

Patent Citations (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US7867491B2 (en) 2007-05-30 2011-01-11 Genexine Co., Ltd. Immunoglobulin fusion proteins
KR101783272B1 (ko) 2008-09-17 2017-09-29 젠코어 인코포레이티드 ＩｇＥ-매개된 장애를 치료하기 위한 신규 조성물 및 방법
KR20120103702A (ko) * 2010-01-19 2012-09-19 에프. 호프만-라 로슈 아게 단백질용 약제학적 제형물
KR102038675B1 (ko) 2018-01-08 2019-10-30 (주)지아이이노베이션 IgE Fc 수용체의 알파 서브유닛의 세포외 도메인, 이를 포함하는 약학적 조성물 및 이의 제조방법
KR102038672B1 (ko) * 2018-01-08 2019-10-30 (주)지아이이노베이션 IgE Fc 수용체의 알파 서브유닛의 세포외 도메인을 포함하는 약학적 조성물
US20200353049A1 (en) * 2018-01-26 2020-11-12 Genentech, Inc. Compositions and methods of use
WO2020097141A1 (en) * 2018-11-07 2020-05-14 Merck Sharp & Dohme Corp. Stable formulations of programmed death receptor 1 (pd-1) antibodies and methods of use thereof

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